Project Summary Alzheimer?s disease (AD) and AD-related dementias have a severe impact on those affected, the healthcare system and the US economy, but currently no treatments exist and a critical need for effective biomarkers persists. The development of ultrasensitive assays such as the single molecule array (SIMOA) platform has facilitated a significant biomarker opportunity that may transform the current landscape and have a large impact on our ability to manage these diseases. Measuring CSF tau is the primary staple for tau biomarkers, but the SIMOA technology has facilitated a significant biomarker opportunity because the presence of very low levels of tau, a microtubule-associated protein that composes the hallmark pathologies of tauopathies (including AD and ADRDs), in plasma can be reliably measured. Continued pursuit of CSF biomarkers is critical, but plasma provides an important opportunity to potentially obtain critical biomarker information through routes that are relatively non-invasive, inexpensive and easy to collect serially in large volumes. Unfortunately, only two SIMOA assays available for measuring plasma tau and neither distinguishes between mild cognitive impairment (MCI) from nondemented control (ND) cases. Thus, there is a critical need for developing novel tau-based assays. Our overall goal is to use our large resource of established, well-characterized monoclonal tau antibodies and newly created and characterized tau monoclonal antibodies to develop a set of novel SIMOA assays for assessing normal and pathological tau in plasma and CSF. We will pursue two independent aims to achieve this goal. In specific aim 1, we will develop tau assays that detect total tau by targeting specific epitopes throughout the protein. Our experience indicates that the forms of tau in biological fluids (such as CSF) are detected with differential sensitivity depending on the antibody, and the best tau region to target in plasma is not defined. Using a step-wise assay development framework, we will validate novel assays using a combination of recombinant tau protein and human plasma and CSF reference samples. Rigorous criteria will be required to further develop an assay, at which point we will compare its performance using plasma and CSF samples from ND, MCI and AD cases. In specific aim 2, we will develop novel tau assays that detect specific pathogenic forms of tau using antibodies that specifically detect conformational or aggregated states of tau, as well as cleaved tau. The presence/abundance of these pathogenic forms of tau in the plasma and utility as biomarkers remains unexplored. We will use the same workflow for assay development as in aim 1 and will test validated assays for their performance with ND, MCI and AD samples. Overall, we propose to use a rigorous set of studies to develop completely novel tau-SIMOA assays for the detection of total tau and pathogenic tau conformations/truncations in human plasma and CSF across the spectrum of ND, MCI and AD. Developing such assays is critical to advance the field toward obtaining assays with high specificity, sensitivity, reproducibility and predictability for identifying tauopathies, such as AD and other ADRD tauopathies.